Numb-Interacting Protein Screening Based On Y[F]XNXXF[Y] Motif And The Numb-Regulated EAAT3 Endocytosis Mechanism

Numb was originally identified as a cell fate determinant involved in the early development of Drosophila. Recent studies uncovered a versatile Numb in membrane protein endocytosis, cell adhesion, cell migration, and ubiquitination of some signaling pathway substrates. Our previous studies have revealed that the conservative YVNHSF motif is critical for NPC1L1 endocytosis through direct binding to adaptor protein Numb. Interestingly, the substitution of the first residue Y for F or of the last residue F for Y does not disrupt the interaction between NPC1L1 and Numb. Y[F]XNXXF[Y] motif is similar to the classical NPXY for Numb mediated vesicular endocytosis but slightly different from it. Y[F]XNXXF[Y] represent a novel signaling sequence different from the classical NPXY motif. To identify whether other proteins may harbor this potential Numb-binding region, we used Y[F]XNXXF[Y] as a query to search protein database and found thousands of candidates matching the criteria. We picked up the proteins which have the functions related to Numb and hold the Y[F]XNXXF[Y] motif in the C terminus, then we got 5 proteins:EAAT3, PXN, RAPGEF5, PRKCA and PIP5K1B. In these 5 candidates, only mutated YVNGGF motif of EAAT3 resulted in a noticeable different localization in cells.EAAT3 is a high-affinity, sodium-dependent glutamate carrier that works at plasma membrane but localizes primarily within the cell. Although it has been known to rapidly translocate between the plasma membrane and the intracellular pool, the molecular details and cellular machinery of EAAT3 trafficking still remain elusive. Here, we reveal a Numb-binding sequence YVNGGF in the C terminus that is critical for the intracellular localization and the apical sorting of EAAT3 in cells. Mutating critical residues Y503, N505 and F508 within the YVNGGF motif or silencing Numb expression by siRNA results in destroyed endocytosis and aberrant distribution of EAAT3. We conclude that Numb is a pivotal adaptor protein imperative for the endocytosis and subcellular localization of EAAT3 through binding to the YVNGGF motif.Taken together, based on finding the interacted proteins of Numb by bioinformatics, we found EAAT3, and explored the influence of Numb on EAAT3 cellular localization and reveals a possible mechanisms of EAAT3 trafficking between the plasma membrane and cytoplasm. It also provides a convenient and effective way to find more Numb interacted proteins.